| ObjectiveBecause rare earth elements and their compounds have the unique physical and chemical properties, they have been widely used in many areas. At the same time, the rare earth elements inevitably bring relevant problems of toxicology because of the extensive application of rare earth elements. Rare earth neodymium oxide (Nd2O3) as a light rare earth oxides, is widely applied in industrial materials world wide. After the eighties of the last century,metal neodymium as the raw material of a new generation of magnetic material NdFeB come out has been applied in many fields and demand for metals has increased dramatically. With the continuous expansion of the metal neodymium application market, and its production scale is developing rapidly. As a raw material for the production of metal neodymium aluminum the amount will also increases, contacting neodymium oxide occupational groups will also increase. A large numbers of professionals who are in contact with xenobiotics rare earth neodymium oxide dust will increase andwhat damage will occur to their health particularly on the respiratory system and the extent of damage mechanism is still not clear. Therefore, we present experimental study, in order to explore the effect and mechanism of rare earth neodymium oxide induce lung injury, provide a scientific basis for the population exposed to safety exposure and workplace occupational exposure limits developed, protect the safety and health of workers in the working process, promote the development of China’s economy and the degree of social civilization.Methods1. Rats were randomly allocated into 2 groups:control (PBS) and Nd2O3 (100 mg/kgin PBS) intratrachealinstillation. Rats were sacrificed at 5 different time points:day 3,7,14,21 and 28 post-instillation.2. Gather rat bronchoalveolar lavage fluid (BALF), analysis of cellular components in BALF, determination of total protein content in BALF, the content of MCP-1, M1P-1α, ICAM-1, IL-1β, TNF-a and IL-6were measured by ELISA methods in BALF.3. Collect rat lung tissue samples, take some specimens with HE staining, after staining pathological changes in lung tissue was observed by light microscope; A portion specimens Sirius red staining, dyeing to analysis lung tissue collagen fiber changes with the Nikon Eclipse E800 polarizing microscope and Image-Pro Plus Version 4.5 forwindows TM image analysis system.4. The rat lung tissue proteins was extracted by Western blotting and non-radioactive gel shift assay EMSA (Electrophoretic Mobility Shift Assay, EMSA) assay rat lung tissue NF-κB p65, p-NFκB p65, p-IKK beta and NF-κB expression.5. To culture Cell NR8383, MTT methods was used to assay the toxic effects of rare earth neodymium oxide; Cells ultrastructural changes and apoptosis were observed by transmission electron microscopy and flow cytometry; The content of MCP-1, MIP-1α, ICAM-1, IL-1β, TNF-a and IL-6 on NR8383 cellswere measured by ELISA methods.6. The infected cell protein was extracted, expressions of p65 p-IκKβ, p-NFκB and Caspase-3 were determined by Western blot.Results1. Weight and lung organ coefficient changes in ratsRats exposed section 3,7,14,21 and 28d, weight were lower than the control group, the difference was statistically significant (P<0.05); The rat lung coefficient were higher control group (P<0.05), suggests neodymium oxide dust-induced lung inflammation causing congestion, edema, or tissue in rats.2. Dust stained lung tissue of rats with type Ⅰ, Ⅲ collagen area percentage changesCompared with the control group, the Ⅰ collagen area percentage of each point dust rats lung was observed in varying degrees of increase;Article 7,14,21 and 28 days the difference was statistically significant (P<0.05). With the extension of dust time, Ⅰ type collagen area percentage gradually increased. Article 28d, Ⅰ type collagen area percentage increased by 1 percent.Compared with the control group, the Ⅲ collagen area percentage of each point dust rats lung was observed in varying degrees of increase; In addition 3d, other groups compared to control group, differences were statistically significant (P<0.05); With the extension of stained dust of time, the percentage area of collagen type Ⅲ tended to increase (P<0.05).3. Determination of cytokine levels in BALF of rats exposedCompared with the control group, the expression of IL-1 and TNF-a was significantly increased in the rats BALF of neodymium oxide dust group (P<0.05); And the expression of IL-6 was increased, the difference was not statistically significant (P> 0.05).Compared with the control group, the expression levels of inflammatory chemokines (MCP-1, MIP-1α and ICAM-1) showed a rising trend in rats BALF of neodymium oxide dust. The expression level of MCP-1 and MIP-1α significantly increase in the only first 28 days (P<0.05).4. Contaminated Dust rats NF-κB-associated protein expressionNeodymium oxide dust dye group, the lung tissue p-NF-κB P65 protein expression increased, while the lung tissue NF-κB P65 total protein level has not changed; On 14 days, the expression levels of p-IKKβ and p-NF-κB P65 peaked in the lung tissue, on day 28 showed a downward trend. The expression of p-NF-κB P65, NF-κB P65 and p-IKKβ not observed changes in the rat lung tissue of control group. NF-κB P50/P65 nuclear translocation of rat lung tissue is induced, and Western blot assay results are consistent. Migration NF-κB P50/P65 increase in the first three days, 14 days up to the peak, began to decline in the first 21 days.5. The toxic effects of rare earth neodymium oxide particles on NR8383 cellsNR8383 cells were exposured with 12hours,24 hours and 48 hours by the suspension of the rare earth Nd2O3 dust,inhibitory rate in the control group compared with 1.56,3.125,6.25μg/mL each exposure group, between each other was not statistically significant (P> 0.05);With the increasing of exposure dose, cell inhibition rate increased(R2=0.959,0.948,0.943). NR8383 cells were exposed to a concentration of 50,100,200μg/mL, significant difference between the exposure time (F=9.54, P<0.05; F=17.596,P<0.05; F=9.332, P<0.05); Rare earth Nd2O3 dust acting on NR8383 cells, cytotoxic was dose-response relationship and rendering time-effect relationship.6. The effect of NR8383 apoptosis in different concentrations of rare earth neodymium oxide particle exposureNR8383 cells were exposured with 24 hours by the suspension of the rare earth Nd2O3 dust, percentage of apoptotic cells in the early stage of each exposure group has showed different degrees of increase. The percentage of apoptotic cells in the early stage of 6.25 and 12.5μg/mL group compared with control group, the difference was statistically significant (P<0.05). The percentage of apoptotic cells in the early stage of 12.5μg/mL group was 7.2 times that of the control group. In 0-25ug/mL concentration range, with increasing exposure concentration, percentage of apoptotic cells in the late stage(1.8%、2.4%、2.6%、3.8%、4.1%) was increasing. The percentage of apoptotic cells in the early stage of 12.5、25 and 50μg/mL group compared with control group, the difference was statistically significant (P<0.05). When 50μg/mL, The percentage of apoptotic cells began to decline.7. The effects of different concentrations of rare earth neodymium oxide particles stimulate the secretion of IL-1β, TNF-α, IL-8, MCP-1, MIP-1α, TGF-β1 on NR8383 cell supernatantsRare earth neodymium oxide exposure 24h,the secretion of IL-1β, TNF-α, IL-8, MCP-1, MIP-1α and TGF-β1 in exposed groups NR8383 cells were higher than the negative control group, the difference was statistically significant (P<0.05). And the secretion of IL-1β, TNF-α, IL-8, MCP-1, MIP-1α and TGF-β1 in exposed groups NR8383 cells with increasing exposure dose showing a rising trend(R2=0.986,0.923, 0.922,0.990,0.985,0.984). Different exposure group difference was statistically significant (P<0.05).8. Correlation analysis of cytotoxic and cytokinesRare earth neodymium oxide exposure 24h, inhibition rate with the content of TNF-α, IL-1β, IL-8, MCP-1, MIP-1α and TGF-β1 was positively correlated (P<0.01); The correlation coefficients of inhibition rate with the content of TNF-α, IL-1β, IL-8, MCP-1, MIP-1α and TGF-β1 were 0.912,0.932,0.933,0.935,0.943 and 0.962.9. The expression of Caspase-3, p-NF-κB p65 and p-IκKβ on NR8383 cellsRare earth neodymium oxide exposure 24h, the expression of p-NFκB p65, p-Ikκβ higher than the negative control group, and the difference was statistically significant at the concentration of 12.5,25 and 50μg/mL (P<0.05). The expression of Caspase-3 higher than the negative control group, and the difference was statistically significant (P<0.05).10. Correlation analysis of cytokines and NF-κB-associated proteinRare earth neodymium oxide exposure 24h, NF-κB-associated protein(p-NF-κB p65, p-IκKβ) with the content of TNF-α, IL-1β, IL-8, MCP-1, MIP-1α and TGF-β1 was positively correlated (P<0.01); The correlation coefficients of p-NF-κB p65 with the content of IL-1β, IL-8, TNF-α, MCP-1, MIP-1α and TGF-β1 were 0.484,0.359, 0.539,0.527,0.535 and 0.598; The correlation coefficients of p-IκKβ with the content of IL-1β, IL-8, TNF-α, MCP-1, MIP-1 aand TGF-β1 were 0.326,0.299,0.406,0.397, 0.407 and 0.391.11. Correlation analysis of cytokines and Caspase-3Rare earth neodymium oxide exposure 24h, Caspase-3 with the content of TNF-α, IL-1β, IL-8, MCP-1, MIP-1α and TGF-β1 was positively correlated (P<0.01); The correlation coefficients of Caspase-3 with the content of IL-1β, IL-8, TNF-α, MCP-1, MIP-1α and TGF-β1 were 0.177,0.121,0.300,0.258,0.300 and 0.277.Conclusions1. Rare Earth Nd2O3 particulate matter into the lung tissue of rats through non indiscretion way, can be induced acute lung injury. Inflammatory injury is the main performance in the early stage, and fibroblasts nodules may be formed in the late stage.2. In the rare earth neodymium oxide particles induced acute lung injury of rats,the expression of pro-inflammatory cytokines, anti-inflammatory cytokines and protein was significantly increased. NF-κB from the cytoplasm to the nucleus transfer and was activated. NF-κB inflammatory pathway was activated, showed that NF-κB in the rare earth neodymium oxide particles induced lung injury has an important role.3. The content of chemokines cytokines and inflammatory cytokines in NR8383 cell supernatants was significantly increased. The expression of p-IKKβ and p-NF-κBp65 in NR8383 cell was significantly increased, showed that NF-κB signaling pathway is activated.4. Degree of apoptosis of alveolar macrophages with the expression level of inflammatory cytokines and chemokines and Caspase-3 temperature increased. Inflammatory cytokines positively correlated with Caspase-3, TNF-α/TNFR/ NF-κB signaling pathway on apoptosis of alveolar macrophages may have a promoting role.5. Alveolar macrophages secrete inflammatory cytokines, the expression level of p-NF-κBP65, p-IKκβ protein, levels of apoptosis in alveolar macrophages and their interaction relationships, showed that NF-κB signaling pathway, alveolar macrophage apoptosis starting is an important mechanism of rare earth neodymium oxide particle-induced lung injury. |
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